MATERIALS AND METHODS

TRIC-B-knockout mice

TRIC-B-knockout mice of the C57BL6J and 129 strain genetic backgrounds were
generated and genotyped as described previously
(Yazawa et al., 2007).
Haematological assessment was carried out using a blood gas analyser (model
GASTAT-601, Techno Medica, Japan), glucose tester and lactate meter (Glutest
Ace and Lactate Pro, Arkray, Japan). Mouse experiments were conducted with the
approval of the Animal Research Committee at Kyoto University, according to
the regulations regarding animal experimentation at Kyoto University.

Phospholipid analysis

The lung was cut into small pieces and digested in PBS-containing 0.5 mg/ml
collagenase (Nitta Gelatin, Japan) at 37°C for 30 minutes. The digested
tissue solution was centrifuged (700 g for 10 minutes), and
the supernatant was recovered as the interstitial fraction. Total lipids in
the fraction were extracted with a chloroform-methanol mixed solvent, and
phospholipids were quantified by conventional colourimetric determination
(Fiske and SubbaRow, 1925). For
further analysis of phospholipid components
(Taguchi et al., 2007), after
adding 1-heptadecanoyl-sn-glycero-3-phosphocholine (17:0-LPC, Avanti Polar
Lipids) as an internal standard, lipids were extracted from the interstitial
fraction and subjected to liquid chromatography-electrospray ionisation mass
spectrometry/tandem mass spectrometry (LC-ESIMS/MS) analysis using the 4000
Q-Trap Quadrupole Linear Ion Trap Hybrid Mass Spectrometer (Applied
Biosystems) equipped with Acquity ultra-performance liquid chromatography
(Waters). Lipid samples were separated using a reverse-phase C18 column, and
phospholipid fractions were injected into the mass spectrometer using an
autosampler.

RESULTS

Respiratory failure in TRIC-B-knockout neonates

We recently generated TRIC-B-knockout mice by gene targeting
(Yazawa et al., 2007).
Heterozygous mice were indistinguishable from wild-type littermates in growth,
development and reproduction. By crossing the heterozygous mice,
TRIC-B-knockout neonates were delivered at the expected Mendelian frequency,
although they were marginally smaller in body size (data not shown). The
knockout neonates wriggled their bodies and gasped for breath, and died
shortly after birth (Fig. 1A).
Respiratory gas exchange is normally initiated at birth, when the mice turn
pink and rapidly inflate their lungs, as indicated by the appearance of white
patches on the thorax. By contrast, although TRIC-B-knockout mice initiated
normal respiratory efforts, they failed to inflate their lungs, remaining
cyanotic and succumbing within an hour. In accordance with this observation,
our haematological assessment detected insufficient plasma O2
pressure, elevated CO2 pressure and reduced pH in the mutant
neonates (Table 1). Therefore,
TRIC-B-knockout neonates die due to severe hypoxia and acidosis resulting from
respiratory failure.

Although the lungs from TRIC-B-knockout neonates showed normal wet-weight
levels (data not shown), the tissues were deflated, the intra-alveolar septa
remained thick and air spaces were diminished
(Fig. 1B). Histological
observations confirmed atelectasis neonatorum in the mutant neonates
(Fig. 1C). In the wild-type
lungs stained with Toluidine Blue, pulmonary alveoli were well-organised by
embryonic day (E) 17.5, and there were many black dots indicative of
surfactant protein aggregates in the alveolar spaces. By contrast, although
the TRIC-B-knockout lungs retained the surfactant aggregates, they did not
form organised alveoli even at the neonatal stage. Therefore, alveolar
hypoplasia seems to be the major cause of death in the mutant neonates.

TRIC channel subtypes in neonatal tissues and alveolar epithelial marker
proteins in TRIC-B-knockout mice. (A) Immunoblot analysis of TRIC
channels in wild-type and TRIC-B-knockout neonates. Total tissue homogenates
were examined using antibodies specific to TRIC channel subtypes and to the
loading control BiP (HSPA5). (B) Immunoblot analysis of alveolar
epithelial markers in neonatal lungs. Total lung homogenates were examined
using antibodies to type II cell markers (mature SP-B, proSP-C and ABCA3), a
type I marker (AQP5) and to the loading control actin.

TRIC channel subtypes are differentially expressed in all tissues. Although
the mature lungs of adult mice contain moderate levels of both TRIC-A and
TRIC-B channels (Yazawa et al.,
2007), immunoblotting clearly indicated that TRIC-B expression is
dominant over TRIC-A expression in the neonatal lung
(Fig. 2A). In the mutant
neonates, the loss of TRIC-B channels might specifically damage a certain cell
type in the lung.

Our histological analysis also suggested the persistent accumulation of
glycogen-rich cells in the TRIC-B-knockout neonatal lung
(Fig. 1C). To confirm this,
glycogen-rich cells were visualised by metachromasy for Toluidine Blue
staining (Fig. 3A). Indeed, the
proportion of glycogen-rich cells in the alveolar epithelium of the
TRIC-B-knockout lung was more than twofold higher than the control value of
the wild-type lung (Fig. 3B).
The alveolar epithelium is composed of type I and type II cells; the type I
cells are squamous and surround the alveolar spaces, whereas the type II cells
are cylindrical and serve to secrete surfactants. Immature type II cells
excessively preserve sugar to generate glycogen deposits in the cytoplasm, and
during their morphological and functional maturation the stored glycogen is
converted into phospholipids to produce lamellar bodies composed of surfactant
lipids and proteins (Ridsdale and Post,
2004). Therefore, the excess of glycogen-rich cells suggests that
the functional maturation of type II cells is severely interrupted in the
TRIC-B-knockout neonates. However, immunoblot analysis
(Fig. 2B) showed normal
expression of type I (AQP5) and type II [mature SP-B (SFTPB), proSP-C (SFTPC)
and ABCA3] cell markers in the TRIC-B-knockout lungs, and immunohistochemical
analysis (see Fig. S1 in the supplementary material) detected a normal
population of type I and type II marker-positive cells in the mutant alveoli.
These observations indicate that the fundamental cell-fate decision occurs
normally in the alveolar epithelium of TRIC-B-knockout mice.

Based on the poor lamellar bodies detected in TRIC-B-knockout neonates, we
predicted reduced phospholipid contents in the mutant alveolar type II cells
as well as in the alveolar space. To examine phospholipid deposits in
intracellular lamellar bodies, we utilised Sudan Black B to differentially
stain phospholipids and nuclei black and pink, respectively
(Fig. 4A). The area of
black-stained granules in the TRIC-B-knockout lung was significantly smaller
than that in the wild-type lung (Fig.
4B). In order to assess surfactant phospholipids in the alveolar
space, we prepared interstitial extracts from the neonatal lungs by a mild
collagenase treatment. Total phospholipid levels in the interstitial fractions
from TRIC-B-knockout lungs were significantly lower than in those from
wild-type lungs (Fig. 4C).
Therefore, both the biosynthesis and secretion of surfactant phospholipids are
impaired in the mutant type II cells.

To further examine the secreted phospholipid species, the interstitial
fractions were subjected to LC-ESIMS/MS analysis (see Fig. S2A,B in the
supplementary material). To reduce the surface tension at the alveolar
air-liquid interface, alveolar surfactant contains a large amount of
phosphatidylcholine (PC) (∼80% of total phospholipids), and lamellar
bodies contain an abundance of PC species carrying palmitate residues
(Rooney, 1985). Not only the
major 16:0-16:1 and 16:0-16:0 PC species
(Fig. 4D), but also the minor
PC species (see Fig. S2C in the supplementary material), were clearly reduced
in the interstitial fractions from the TRIC-B-knockout lungs.
Phosphatidylglycerol (PG) is the second major phospholipid in lung surfactant,
accounting for ∼10% of the total phospholipids
(Rooney, 1985). Major PG
species, including 16:0-16:1 and 16:0-16:0 PG, were also decreased in the
mutant lung (see Fig. S2D in the supplementary material). Moreover, we
detected lower amounts of phosphatidylethanolamine, phosphatidylserine and
phosphatidylinositol species in the mutant lung (see Fig. S2E in the
supplementary material).

After lamellar bodies are secreted into the alveolar space, the surfactant
undergoes a sequential morphological transformation to tubular myelin, small
vesicles and mono- and multi-layers. The transformation processes depend upon
the phospholipid and protein composition of the secreted lamellar bodies.
Tubular myelin is not essential for alveolar inflation
(Ikegami et al., 2001), but
seems to contribute to the formation of lipid layers at the air-liquid
interface (Hallman, 2004). In
the electron microscopy analysis, tubular myelin structures were frequently
observed in the wild-type neonatal lungs, whereas we could not detect typical
tubular myelin in TRIC-B-knockout neonates (see Fig. S3 in the supplementary
material). Together with the results from the LC-ESIMS/MS analysis, this
ultrastructural abnormality provides further evidence of the insufficient
quantity and deranged composition of the surfactant phospholipids secreted
into the alveolar space of the TRIC-B-knockout lung.

Because TRIC channels support Ca2+ release from the muscle SR,
and alveolar type II cells exhibit morphological and metabolic abnormalities
in TRIC-B-knockout neonates, we next surveyed Ca2+ signalling
defects in the mutant type II cells. When primary alveolar cells were prepared
in culture, type II cells were distinguishable from type I cells by their
appearance: the type I cells were squamous, whereas the type II cells were
cuboidal. This assignment was further confirmed by immunostaining for the
alveolar epithelial markers AQP5 and proSP-C (see Fig. S1C in the
supplementary material). It is well recognised that type II cells gradually
lose their specialised features in primary culture. To minimise this
dedifferentiation and to standardise cellular conditions, cultured type II
cells were subjected to Ca2+ imaging analysis after ∼72
hours.

Fura-2 measurements detected three major defects in cultured
TRIC-B-knockout type II cells. The first striking abnormality was that resting
Ca2+ levels in the mutant cells were significantly lower than those
in wild-type cells in a normal bathing solution. However, Ca2+
removal from the bathing solution reduced cytoplasmic Ca2+ levels
in both the mutant and wild-type type II cells, and similar resting levels
were observed in both under Ca2+-free conditions
(Fig. 5A,B). Several molecular
mechanisms can be proposed for the impressive fluctuation in the resting
Ca2+ level seen in the TRIC-B-knockout cells (see Discussion).

Because the ryanodine receptor agonist caffeine did not evoke detectable
Ca2+ transients, it is likely that type II cells do not contain
intracellular stores equipped with ryanodine receptors (data not shown).
Surfactant secretion from type II cells is stimulated by purinergic
P2Y2 receptor (P2RY2) activation, which results in IP3
receptor-mediated Ca2+ release
(Haller et al., 1998). In
agreement with the previous report, ATP application to cultured type II cells
generated Ca2+ transients. The second phenotype of TRIC-B-knockout
type II cells was impaired agonist-induced Ca2+ transients
(Fig. 5A,C). Because the PAR4
(F2RL3) receptor is also expressed in type II cells
(Ando et al., 2007),
IP3 receptor-mediated Ca2+ release can be induced by a
PAR4 agonist peptide (sequence AYPGKF). TRIC-B-knockout type II cells also
exhibited weakened PAR4 agonist-evoked Ca2+ transients (see Fig.
S4A,B in the supplementary material). It is unlikely that the compromised
IP3 receptor-mediated Ca2+ release was due to defects in
phosphatidylinositol turnover, as no differences were observed in the
expression of signalling proteins between the TRIC-B-knockout and wild-type
lungs (see Fig. S5 in the supplementary material).

The three atypical features in Ca2+ handling described above are
specific to alveolar type II cells in TRIC-B-knockout mice, as we could not
detect any abnormalities in the Ca2+ imaging data from mutant
alveolar type I cells (see Fig. S6 in the supplementary material), cardiac
myocytes (see Fig. S7 in the supplementary material) or embryonic fibroblasts
(see Fig. S8 in the supplementary material). These observations imply that
abnormal Ca2+ handling is closely linked to the morphological and
biochemical defects in TRIC-B-knockout type II cells during perinatal
maturation.

Proposed role of the TRIC-B channel in type II cells. By functioning
as a counter ion channel coupled with IP3 receptor-mediated
Ca2+ release in intracellular stores, TRIC-B channels seem to
stimulate agonist-induced Ca2+ transients and maintain resting
Ca2+ levels in type II cells. Because cellular Ca2+
signals are probably involved in the functional and morphological maturation
of type II cells at the perinatal stage, impaired Ca2+ handling
caused by the TRIC-B deficiency is likely to disturb the maturation processes,
leading to respiration failure.

During the functional maturation of alveolar epithelial cells, type I cells
differentiate from immature type II cells in response to TGFβ/Smad
signalling (Bhaskaran et al.,
2007) and become AQP5 positive
(Williams, 2003), whereas type
II cells start to abundantly synthesise phospholipids and surfactant
associated proteins A to D (SP-A to D; SFTPA to D)
(Rooney et al., 1994). Based
on immunochemical data with alveolar marker proteins
(Fig. 2B and see Fig. S1 in the
supplementary material), TRIC-B channels are not involved in cellular
differentiation of alveolar epithelium. However, insufficient glycogen
breakdown and phospholipid synthesis (Figs
3 and
4) probably result from
compromised Ca2+ handling in TRIC-B-knockout type II cells, as
sugar and lipid metabolisms are controlled by several key
Ca2+-dependent enzymes, including glycogen phosphorylase, pyruvate
dehydrogenase and fatty acid synthase
(Davis and Kauffman, 1986;
Terrand et al., 2001;
Nasser et al., 2004). The
combination of reduced resting Ca2+ levels and insufficient
agonist-evoked Ca2+ transients presumably inactivates these
Ca2+-dependent enzymes, and thereby might inhibit the conversion of
glycogen into phospholipids during the perinatal maturation of TRIC-B-knockout
type II cells. Moreover, abnormal Ca2+ handling is likely to
inhibit the formation of lamellar bodies containing phospholipids and
surfactant proteins (Fig. 3),
as Ca2+-dependent fusion between juvenile lamellar bodies is
required for their morphological maturation
(Dietl and Haller, 2005). The
impaired biosynthesis of phospholipids, together with their poor secretion
(Fig. 4) and atypical
structural features in the alveolar space (see Fig. S3 in the supplementary
material), led us to conclude that these abnormalities combine to produce a
lethal defect in lung inflation in TRIC-B-knockout neonates
(Fig. 6).

Our data further raise the possibility that impaired Ca2+
handling could interrupt Ca2+-dependent kinase/phosphatase
signalling in TRIC-B-knockout type II cells. Calcineurin is a
Ca2+/calmodulin-dependent phosphatase that directly
dephosphorylates nuclear factors of activated T cells (NFATs) in the cytoplasm
to promote their translocation into the nucleus and participation in the
transcriptional regulation of diverse genes. Mutant mice with a pulmonary
epithelium-specific deletion of the calcineurin b1 (Ppp3r1) gene show
respiratory failure immediately after birth, and in the mutant lungs the
expression of SP-A to D and ABCA3 is almost absent. Therefore, the expression
of the type II cell-specific proteins is transcriptionally upregulated under
the control of the calcineurin-NFAT cascade during perinatal maturation
(Dave et al., 2006). By
contrast, normal expression levels of SP-B, proSP-C and ABCA3 were observed in
the TRIC-B-knockout lungs (Fig.
2B), indicating that calcineurin-NFAT signalling is not
significantly affected in the mutant type II cells. Although the roles in lung
development of the Ca2+/calmodulin-dependent kinases, protein
kinase C subtypes and Ca2+-dependent tyrosine kinases are largely
unknown, it is interesting to speculate that insufficient signals from such
Ca2+-dependent kinases might amplify or attenuate the primary
defects that result from the gene ablation in TRIC-B-knockout type II
cells.

Supplementary material

Footnotes

We thank Naoya Onohara for reading the manuscript. This work was supported
in part by research grants from the Ministry of Education,
Culture, Sports, Science and Technology, the
Ministry of Health and Welfare of Japan,
US National Institutes of
Health, the Kurata
Memorial Foundation, the Japan Foundation for
Applied Enzymology and the Novartis
Foundation. Deposited in PMC for release after 12 months.

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